Ritter & Oleson's Colloidal Iron
for Acid Mucosubstances

Solutions

Acetic acid, 2M (12%)

Potassium ferrocyanide, 1% aqueous

Hydrochloric acid, 1N aqueous

Nito & Stoke's colloidal iron suspension.

Schiff's reagent

Working colloidal iron
Colloidal iron suspension	1	vol
Acetic acid, 2M	1	vol

Perls' solution
1% potassium ferrocyanide	86	mL
Hydrochloric acid, 1N	14	mL

Stock sodium acetate
Sodium acetate	4	g
Ethanol, absolute	1	L

Periodic acid
Periodic acid	0.4	g
Stock sodium acetate	5	mL
Distilled water	100	mL

Reducing rinse
Potassium iodide	1	g
Sodium thiosulphate	1	g
Distilled water	20	mL
Ethanol, absolute	30	mL
Hydrochloric acid, 2N	0.5	mL

Sulphite rinse
Distilled water	100	mL
Potassium metabisulphite	0.4	g
Hydrochloric acid, conc.	1	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Dichromate should be avoided.

Method

1. Bring sections to water via xylene and ethanol.
2. Place into the working colloidal iron suspension for 10 minutes.
3. Wash with distilled water.
4. Place into freshly made Perls' solution for 10 minutes.
5. Wash with distilled water.
6. Rinse with 70% ethanol.
7. Oxidise in periodic acid for 5 minutes.
8. Rinse with 70% ethanol.
9. Place in the reducing rinse for 5 minutes.
10. Rinse with 70% ethanol.
11. Place in Schiff's reagent for 1 hour.
12. Wash in sulphite rinses, 3 changes of 1½ minutes each.
13. Wash with running tap water for 5 minutes.
14. Optionally, counterstain with a hemalum such as Mayer's for 1 minute, and blue.
15. Dehydrate with ethanols.
16. Clear with xylene and mount with a resinous medium.

Expected results

• Acid mucopolysaccharides  –  blue
• Neutral mucopolysaccharides  –  red
• Glycogen  –  red
• Nuclei  –  blue

Notes

1. The original method used a commercial colloidal iron preparation. This is still available. Nito & Stokes colloidal iron suspension is also suggested.
2. Since this method depends on the staining of iron compounds with the prussian blue reaction, any hemosiderin present will also be stained. If this is a concern, a control section should be stained which has not been treated with colloidal iron. Material stained blue in both sections should be discounted.
3. A PAS is integral to this method. A simpler PAS procedure would probably be as satisfactory. Acid mucosubstances will be stained blue in contrast to red neutral mucosubstances. However, they are often present as mixtures and the contrast may not be clear.

 

Reference
Humason, G. L., (1967)
Animal tissue techniques, Ed. 2
W. H. Freeman and Company, San Francisco, Ca, USA